Glycosylated antibody

ABSTRACT

The invention relates to a CHO cell-line capable of producing antibody, the cell-line having been co-transfected with a vector capable of expressing the light chain of the antibody and a vector capable of expressing the heavy chain of the antibody wherein the vectors contain independently selectable markers; also included is a CHO cell-line capable of producing a human antibody or an altered antibody, the cell-line having been transfected with a vector capable of expressing the light chain of the antibody and the heavy chain of the antibody; process for the production of antibody using a CHO cell-line and antibody having CHO glycosylation.

[0001] The present invention relates to Chinese hamster ovary (CHO) celllines, to the production of proteins, in particular antibodies from suchcell lines, also to antibodies having CHO glycosylation.

[0002] Antibodies or immunoglobulins are proteinaceous bifunctionalmolecules. One region which is highly variable between differentantibodies is responsible for binding to an antigen (Fab region), forexample the many different infectious agents that the body mayencounter, whilst the second, constant region (or Fc region) isresponsible for binding to the Fc receptors of cells and also activatescomplement. In this way, antibodies represent a vital component of theimmune response of mammals in destroying foreign microorganisms andviruses.

[0003] An antibody molecule is composed of two light chains and twoheavy chains that are held together by interchain disulphide bonds. Eachlight chain is linked to a heavy chain by disulphide bonds and the twoheavy chains are linked to each other by disulphide bonds. Each heavychain has at one end a variable domain followed by a number of constantdomains, and each light chain has a variable domain at one end and aconstant domain at the ocher end. The light chain variable domain isaligned with the variable domain of the heavy chain. The light chainconstant domain is aligned with the first constant domain of the heavychain. The remaining constant domains of the heavy chains are alignedwith each other. The constant domains in the light and heavy chains arenot involved directly in binding the antibody to the antigen.

[0004] The variable domains of each pair of light and heavy chains formthe antigen binding site. They have the same general structure with eachdomain comprising a framework of four regions, whose sequences arerelatively conserved, connected by three complementarity determiningregions (CDRs). The four framework regions largely adopt a beta-sheetconformation and the CDRs form loops connecting, and in some casescomprising part of, she beta-sheet structure. The CDRs are held in closeproximity by the framework regions and, with the CDRs from the otherdomain, contribute to the formation of the antigen binding site.

[0005] The immunisation of an animal with an antigen results in theproduction of different antibodies with different specificities andaffinities. An antiserum obtained from the immunised animal will,therefore, be heterogeneous and contain a pool of antibodies produced bymany different lymphocyte clones. Antibodies thus obtained are referredto as polyclonal antibodies and this polyclonal nature has been a majordrawback in the use of antibodies in diagnostic assays and intherapeutic applications.

[0006] A major step forward occurred in 1975 when Kohler and Milstein(Nature, 1975, 256, 495-497) reported the successful fusion of spleencells from mice immunized with an antigen with cells of a murine myelomaline. The resulting hybrid cells, termed hybridomas, have the propertiesof antibody production derived from spleen cells and of continous growthderived from the myeloma cells. Each hybridoma synthesizes and secretesa single antibody to a particular determinant of the original antigen.To ensure that all cells in a culture are identical, i.e. that theycontain the genetic information required for the synthesis of a uniqueantibody species, the hybridomas resulting from cell fusion are clonedand subcloned. In this way, the cloned hybridomas produce homogeneous ormonoclonal antibodies.

[0007] The advantages of hybridoma technology are profound. Because manyhybrids arising from each spleen are screened for their potential toproduce antibodies to the antigen of interest and only a few areselected, it is possible to immunize with impure antigens and yet obtainspecific antibodies. The immortality of the cell line assures that anunlimited supply of a homogeneous, well-characterised antibody isavailable for use in a variety of applications including in particulardiagnosis and immunotherapy of pathological disorders. Unfortunately,the usefulness of such monoclonal antibodies in a clinical setting canbe severely hampered by the development of human anti-mouseantibodies—an anti-globulin response—which may interfere with therapy orcause allergic or immune complex hypersensitivity.

[0008] When, for example, murine (or ratine) monoclonal antibodies areused in human therapy, the induction of an human anti-mouse antibodyresponse is due to the murine origin of the constant domains and fourframework regions. This problem has therefore been addressed by thedevelopment of antibodies of two basic types. The first type, referredto as chimeric antibodies, is where the murine constant domains only arereplaced by equivalent domains of human origin (Morrison et al.P.N.A.S., 1984, 81, 6851-6855; Boulianne et al, Nature, 1985, 314,268-270; and Neuberger et al, Nature, 1985, 314, 268-270). The secondtype is where the murine constant domains and the murine frameworkregions are all replaced by equivalent domains and regions of humanorigin. This second type of antibody is referred to as a humanised orCDR-grafted antibody (Jones et al, Nature, 1986, 321, 522-525; andRiechmann et al, Nature, 1988, 332, 323-327). A human antibody would ofcourse avoid the need for “humanisation”, however cell lines whichsecrets human antibodies are very unstable and have generally provenunsuitable for commercial scale production.

[0009] To generate sufficient quantities of antibody for full clinicaluse it is desirable to employ an efficient recombinant expressionsystem. Since myeloma cells represent a natural host specialized forantibody production and secretion, cell lines derived from these havebeen used for the expression of recombinant antibodies. Often, complexvector design, based around immunoglobulin gene regulatory elements, isrequired, and final expression levels have been reported which arehighly variable (Winter et al, Nature, 1988, 332, 323-327; Weidle et al,Gene, 1987, 60, 205-216; Nakatani et al, Bio/Technology, 1989, 7,805-810; and Gillies at al, Bio/Technology, 1989, 7, 799-804).

[0010] An alternative mammalian expression system is that offered by theuse of dihydrofolate reductase (dhfr) deficient Chinese hamster ovary(CHO) cells. The use of these cells has enabled the production of largequantities of several therapeutic proteins for research and clinical use(Kaufman et al, Mol, Cell, Biol, 1985, 5, 1750-1759; and Zettlmeissl etal, Bio/Technology, 1987, 5, 720-725). There are, however, very fewinstances of the use of these cells for the expression of antibodies andthe levels of expression of murine antibodies reported to date arelow—of the order of 0.01-0.1 μg/ml (Weidle et al, Gene, 1987, 51, 21-29;and Feys et al, Int. J. Cancer, 1988, 2, 26-27).

[0011] A process has now been developed that enables the balancedexpression of the light and heavy chains of an antibody from CHO cells.Balanced expression is desirable given that the light and heavy chainsare linked together in the antibody molecule in equimolar proportions.This process allows the antibody to be obtained in functional form andto be secreted in good yields. Thus the process enables sufficientquantities of functional antibody to be obtained for use in theimmunotherapy of pathological disorders.

[0012] The invention therefore provides a CHO-cell line capable ofproducing antibody, the cell line having been co-transfected with avector capable of expressing the light chain of the antibody and avector capable of expressing the heavy chain of the antibody wherein thevectors contain independently selectable markers.

[0013] The present invention further provides a CHO cell line capable ofproducing a human antibody or an altered antibody, the cell line havingbeen co-transfected with a vector containing cDNA encoding the lightchain of the antibody and a vector containing cDNA encoding the heavychain of the antibody said vectors capable of expressing the light andheavy chains of the antibody. The vectors may advantageously containindependently selectable markers. Hereafter, reference to the markersincludes the singular and vice versa.

[0014] The cell line of the present invention is capable of producingall kinds of antibodies chat generally comprise equimolar proportions oflight and heavy chains. The invention therefore includes humanantibodies wherein the amino acid sequences of the heavy and lightchains are homologous with those sequences of antibodies produced byhuman lymphocytes in vivo or in vitro by hybridomas. Also included inthe invention are altered antibodies such as hybrid antibodies in whichthe heavy and light chains are homologous to a natural antibody but arecombined in a way that would not occur naturally. For example, abispecific antibody has antigen binding sites specific to more than oneantigen. The constant region of the antibody-may relate to one or otherof the antigen binding regions or may be from a further antibody.Altered antibodies, such as chimaeric antibodies have variable regionsfrom one antibody and constant regions from another. Thus, chimaericantibodies may be species/species chimaeras or class/class chimaeras.Such chimaeric antibodies may have one or more further modifications toimprove antigen binding ability or to alter effector functioning.Another form of altered antibody is a humanised or CDR-grafted antibodyincluding a composite antibody, wherein parts of the hypervariableregions in additon to the CDRs are transferred to the human framework.Additional amino acids in the framework or constant regions of suchantibodies may be altered. Included in the definition of alteredantibody are Fab fragments which are roughly equivalent to the Y branchportions of the heavy and light chains; these may be included incompletefragments or fragments including part of the Fc region. Thus, within thescope of the invention is included, any altered antibody in which theamino acid sequence is not one which exists in nature.

[0015] The cell line of the invention is preferentially employed for theproduction of altered antibodies most preferably chimaeric antibodies orCDR-grafted antibodies. Particular examples of these include antibodiesagainst T cell markers such as CD2, CD3, CD4, CD5, CD7, CD8, CD11a,CD11b, CD18, CD19, CD25, CD45 and CDw52 and especially CDR graftedantibodies against the CDw52 antigen, such as Campath-1H (Campath is aTrademark of the Wellcome Foundation Ltd) described in EP 328404 Furtherexamples include CDR-grafted antibodies against various cancer cellmarker antigens such as CD33 and CD38.

[0016] After co-transfection into recipient CHO cells, the resultingcolonies may be selected using both markers. Colonies exhibiting thedual phenotype are generally capable of co-expressing both the light andheavy chains. The selectable markers may or may not be of a dominantnature. Examples of selectable markers for use co-transfection includeadenosine deaminase (Kaufman et al, P.N.A.S., 1989, 83, 3136-40)asparagine synthetase (Cartier et al, Mol, Cell Biol., 1987, 7,1623-28), E. coli trpB gene and Salmonella hisD gene (Hartman et al,P.N.A.S., 1988, 85, 8407-51), M2 mouse ribonucleotide reductase(Thelander et al, EMBO J, 1989, 8, 2475-79), human multidrug resistancegene (Kane et al, Gene, 1989, 84, 439-446), glutamine synthetase(Bebbington et al, DNA Cloning, Vol III, 1987, Ed. D. M. Glover,163-188, IRL Press), xanthine guanine phosphoribosyl transferase (gpt)(Mulligan et al, Science, 1980, 209, 1422-27), hygromycin B (Santerre etal, Gene, 1984, 30, 147-156), neomycin gene (Southern et Al, J. Mol.Appl. Genet., 1982, 1, 327-341), and dihydrofolate reductase (Subramaniet al, Mol, Cell Biol., 1981, 1, 854-864). One particularly preferredselectable marker is dhfr which is usually employed with a parental CHOcell line of the dhfr phenotype (Urlaub et al, P.N.A.S., 1980, 77,4216-4220). Successfully co-transfected CHO cells will possess the dhfr⁺phenotype and can readily be selected by culturing the colonies on mediadevoid of thymidine and hypoxanthine and optionally containingmethotrexate (MTX). A preferred selectable marker for use with the otherof the vectors is a dominant resistance marker, such as neomycin (neo).CHO cells successfully transfected with this marker can readily beselected by culturing the colonies on media containing the antibiotic,G418, otherwise known as Geneticin.

[0017] A second preferred system of selection and amplification isprovided by the glutamine synthetase selectable marker or (GS system)which is described in WO87/04462. CHO cells which have been successfullytransfected with the gene encoding the GS enzyme and the desiredantibody gene can be selected by culturing colonies in media devoid ofglutamine as described in PCT published application number WO87/04462.

[0018] At lease one of the selectable markers preferably also providesthe basis upon which the genes encoding the light and heavy chains maybe amplified. In co-transfection of a CHO cell line, the vector DNAs areoften integrated into the chromosome of the cell at the same locus.Thus, the use of only one of the selectable markers as the basis foramplification normally results in a parallel increase in the copy numberof both genes. One particularly preferred selectable marker for use inthis way is dhfr which enables the desired amplification to be obtainedthrough the use of increasing concentrations of MTX. A second preferredselectable marker is GS which allows amplification by the addition ofmethionine sulphoximine (MSX).

[0019] The selectable markers are of course under the control ofregulatory elements of DNA so as to provide for their expression. In thecase of the use of dhfr as a selectable marker, the regulatory elementsare preferably of a viral source, such as from DNA tumour viruses.Particularly preferred are the use of an SV40 or adenovirus major latepromoter. It is particularly advantageous in this regard to remove theenhancer element from the promoter thus effectively “crippling” it. Thismodification allows for increased levels of gene amplification at eachconcentration of methotrexate selection than would otherwise occur if astrong promoter was used. In the case of the use of neo as a selectablemarker, an example of a suitable promoter is the mouse metallothioneinpromoter.

[0020] The light and heavy chain genes may constitute genomic DNA or, rA preferably, cDNA, and are cloned using procedures known in the art(Molecular Cloning: A Laboratory Manual, Second Edition, Maniatis et al,Cold Spring Harbor). The genes are also under the control of regulatoryelements of DNA so as to provide for their expression. The use of thesame regulatory elements for both chains is preferred so that theirexpression is substantially balanced. The regulatory elements may be ofviral origin and examples include those mentioned above in conjunctionwith the expression of dhfr as a selectable marker. Another example isthe use of the β-actin promoter and cognate β-actin polyadenylationsignal.

[0021] One or both of the vectors may also contain an SV40 origin ofreplication to allow for the vector constructs to be checked by rapidtransient assay.

[0022] Construction of the expression vectors may be carried out inaccordance with procedures known in the art (Molecular Cloning: ALaboratory Manual, Second Edition, Maniatis et al, Cold Spring Harbor).

[0023] Co-transfection of the CHO cell line with the expression vectorsmay be carried out simply by using equimolar quantities of both vectorsand standard transfection procedures, such as calcium phosphateprecipitation or lipofectin. Selection of the desired co-transfectedcell line may be carried out in accordance with standard proceduresknown for the particular selectable markers.

[0024] The present invention also provides a process for the productionof an antibody which comprises culturing a CHO cell line of the presentinvention. Culture of the CHO cell line may be carried out inserum-containing or preferably serum and protein free media. In onepreferred instance where the CHO cell line is a dhfr⁺ transformant, themedium preferably lacks hypoxanthine and/or thymidine and optionallycontains MTX. Where a selectable marker is glutamine synthetase themedium preferably lacks glutamine and optionally contains MSX.Expression of both chains in substantially equimolar proportions enablesoptimum yields of functional antibody to be obtained. The two chainsassemble within the cell and are then secreted into the culture mediumas functional antibody. The resulting antibody may be purified andformulated in accordance with standard procedures.

[0025] Antibodies are glycoproteins containing between 3 and 12%carbohydrate. The carbohydrate units are transferred to acceptor siteson the antibody chains after the heavy and light chains have combined.The major carbohydrate units are attached to amino acid residues of theconstant region of the antibody. Carbohydrate is also known to attach tothe antigen binding sites of some antibodies and may affect theantibody-binding characteristics by limiting access of the antigen tothe antibody binding site. There are a number of roles associated withthe carbohydrate units. They may affect overall solubility and the rateof catabolism of the antibody. It is als known that carbohydrate isnecessary for cellular secretion of some antibody chains. It has beendemonstrated that glycosylation of the constant region plays a vitalrole in the effector functioning of an antibody; without thisglycosylation in its correct configuration, the antibody may be able tobind to the antigen but may not be able to bind for example tomacrophages, helper and suppressor cells or complement, to carry out itsrole of blocking or lysing the cell to which it is bound.

[0026] It has now been found that antibody glycosylated by CHO cellsmaintains antigen binding capability and effector functionality. Thishas been demonstrated in in vitro complement lysis assays and in vivo ina human patient.

[0027] The invention therefore provides an antibody having CHOglycosylation. Such antibodies may be natural, such as human antibodies,altered antibodies for example hybrid antibodies or bispecificantibodies, chimaeric or CDR-grafted antibodies, including Fabfragments.

[0028] The CHO glycosylation may be associated with the antigen bindingsite or other parts of the variable domain. It may alternatively oradditionally be associated with the constant region. The glycosylatedantibody is prepared by expression of the antibody genes in a suitablyengineered CHO cell followed by recovery and if necessary, purificationof the antibody from the cell culture medium.

[0029] CHO glycosylated antibodies are useful in medical therapy forcreating numerous human disorders, generally as immunosuppressives moreparticularly for example T-cell mediated disorders including severevasculitis, rheumatoid arthritis, systemic lupis, also autoimmunedisorders such as multiple sclerosis, graft vs host disease, psoriarsis,juvenile onset diabetes, Sjogrens' disease, thyroid disease, myastheniagravis, transplant rejection and asthma. These antibodies are alsouseful in treating cancer such as Non-Hodgkins lymphoma, multiplemyeloma, and infectious diseases such as HIV and herpes.

[0030] The invention therefore provides the use of CHO glycosylatedantibodies in the manufacture of a medicament for the treatment of anyof the aforementioned disorders. Also provided is a method of treating ahuman being having any such a disorder comprising administering to saidindividual a therapeutically effective amount of a CHO glycosylatedantibody.

[0031] The dosages of such antibodies will vary with the condition beingtreated and the recipient of the treatment, but will be in the range 1to about 100 mg for an adult patient preferably 1-10 mg usuallyadministered daily for a period between 1 and 30 days. A two part dosingregime may be preferable wherein 1-5 mg are administered for 5-10 daysfollowed by 6-15 mg for a further 5-10 days.

[0032] Also included within the invention are formulations containingCHO glycosylated antibody. Such formulations preferably include, inaddition to antibody, a physiologically acceptable diluent or carrierpossibly in admixture with other agents such as other antibodies r anantibiotic. Suitable carriers include but are not limited tophysiological saline, phosphate buffered saline, phosphate bufferedsaline glucose and buffered saline. Alternatively, the antibody may belyophilised (freeze dried) and reconstituted for use when needed by theaddition of an aqueous buffered solution as described above. Routes ofadministration are routinely parenteral including intravenous,intramuscular, subcutaneous and intraperitoneal injection or delivery.

[0033] The accompanying drawings show:

[0034]FIG. 1

[0035] (a) the pLD9 construct containing expression cassettes for the‘crippled’ dhfr selection/amplification marker and the Campath-1H lightchain cDNA. The small box with the dashed arrow is the weakened SV40promoter; the larger dotted box with an arrow is the β-actin promoter;polyA refers to respectively sourced polyadenylation and terminationsignals; the small box with ori contains the SV40 origin of replication;

[0036] (b) the pNH316 construct containing expression cassettes for theneomycin selection marker and the Campath-1H heavy chain cDNA. The boxwith an arrow and MT refers to the mouse metallothionein promoter.Restriction sites indicated are: —H, HindIII; Bg, Bg1II; B, BamHI; R1,EcOR1.

[0037]FIG. 2

[0038] Comparative determinations of the rate of Campath-1H synthesis inconfluent A39 cells over 4 consecutive days. Following the [³⁵S]methionine pulse period, equal aliquots of cells (C) and culture medium(M) were immuno-precipitated and separated by SDS-PAGE. The position ofthe Campath-1H heavy and light chains are indicated (H and L arrows).There was some loss of material for the day 3 cell sample.

[0039]FIG. 3

[0040] A pulse-chase experiment co determine the rate of secretion anddistribution of radiolabelled Campath-1H in A39 cells. Confluent cellswere pulsed with [³⁵S] methionine for 6 hours, then fresh mediumcontaining an excess of unlabelled methionine was added. Equal aliquotsof cells and culture medium were taken at the indicated time points (inhours following the end of the pulse period) and treated as described inthe legend of FIG. 2. The samples for the 48 and 72 hour medium timepoints were run on a different gel to the 6 and 24 hour points, and thetracks are only lined up relative to the position of the heavy (H)chain.

[0041]FIG. 4

[0042] Shows growth of ClH 3D11 44 in WCM5 (protein-free medium) in a 1litre fermenter measured as cell count/ml over 90 days.

[0043]FIG. 5

[0044] Shows antibody production from C1H 3D 44 cells in WCM5 in a 1litre fermenter measured as micrograms of antibody/ml over 80 days.

[0045] The following Examples are provided purely for illustration ofthe present invention.

EXAMPLE 1 Cloning of the Heavy and Light Chain cDNAs for Campath-1H

[0046] The complementarity determining regions from the rat Campath-1Gmonoclonal were originally grafted directly into genomic human heavy andlight chain frameworks (Winter et al, Nature, 1988, 322, 323-327).

[0047] These constructs were engineered for expression in the myelomacell line YO and resulted in yields f Campath-1H of up to 5 μg/mlfollowing 10-14 days in culture (Hale et al, Tissue Antigens, 1990, 35,118-127 and Winter et al, Nature, 1988, 322, 323-327). The myeloma cellline TF57 (Hale et al, ibid,) was used to generate size selected cDNAfractions of 0.9-1.2 kb and 1.4-1.7 kb for the light and heavy chaincDNAs respectively. These were used to make EcOR1 Tinkered cDNAlibraries in λgt10. All procedures were as described by Huynh et al (DNACloning, Vol I: A Practical Approach, 1984, Glover, D(Editor), IRLPress, oxford). The libraries were screened using [³²P] nick translatedprobes specific for the variable regions to isolate full length cDNAclones. For the light chain cDNA, the 5′ untranslated leader was removedup to position −32 using Bal-31 exonuclease and a HindIII linker added.For the 3′ end, use was made of a unique. SacI site 47 bp upstream ofthe stop codon. A SacI-HindIII oligonucle tide pair was used toregenerate this sequence and position the HindIII site immediately afterthe stop codon. For the 5′ end of the heavy chain cDNA, the unique NcoIsite overlapping the ATG start codon was used to re-build a 29 bpuntranslated leader, identical to that of the light chain, using aHindIII-NcoI oligonucleotide pair. At the 3′ end, the unique NaeI site12 bp downstream of the stop codon was converted into a HindIII siteusing linkers.

EXAMPLE 2 Construction of Vectors

[0048] The human β-actin promoter was excised from pHβAPr-3-neo (whichcorresponds to pHβAPr-1-neo (Gunning et al P.N.A.S., 1987, 84, 483-35)except that the SV40 polyadenylation/termination signal has beenreplaced with the respective human β-actin signals) as a 2860 bpPvuII-HindIII fragment, in which the PvuII site was subsequentlyconverted to a Bg1II site using linkers. To isolate the human β-actinpolyadenylation and termination signals from pHβAPr-3-neo, an SphI site1.4 kb downstream of the unique HindIII site was converted to a BamHIsite using linkers. The basal dhfr vector called p104, was constructedas follows. The SphI site at position −128 in the SV40 promoter in pSV2dhfr (Subramani et al, Mol, Cell, Biol, 1981, 1, 854-864) was convertedinto a SalI site to remove all enhancer elements from the promoter. Theweakened dhfr expression unit was then subcloned as a SalI-BamHIfragment into the homologous sites in pSVOd (Mellon et al, Cell, 1981,27, 279-288).

[0049] To construct pLD9, the p104 vector was digested with BamHI,phosphatased, and ligated with three other fragments consisting of theBglII-HindIII β-actin promoter, the HindIII Campath-1H light chain cDNAand the HindIII-BamHI β-actin polyA/termination signals. To constructpNH316, the construct pdBPV-MMTneo (Law, et al, Mol, Cell, Biol., 1983,3, 2110-2115) was digested with BamHI, phosphatased, and the fragmentcontaining the neomycin gene isolated following separation on an agarosegel. This was ligated to the two β-actin fragments and the Campath-1Hheavy chain cDNA. The constructs, pLD9 and pNH316 are depicted in FIG.1.

EXAMPLE 3 Expression of Campath-1H in CHO Cells

[0050] The dhfr CHO cell line DUK-B11 (Urlaub et al, P.N.A.S., 1980, 77,4216-4220) was grown in Iscove's MEM supplemented with 10% fetal bovineserum, and 4 μg/ml each of hypoxanthine and thymidine. 10 μg of pLD9 andpNH316 was co-precipitated onto cells using the calcium phosphatemethod, (Gorman et al, DNA Cloning, 1985, Vol II, 143-190, AcademicPress, N.Y.) and selected for the double phenotype of dhfr⁺/neoresistance by using the medium above except that 10% dialysed serum wasused, the hypoxanthine/thymidine were omitted, and G418 (Gibco) wasincluded at 500 μg/ml. In some experiments MTX was included directly inthe first round selection for dhfr⁺ transformants. Several hundredresistant colonies were pooled and assayed for the production ofCampath-1H antibody in the culture medium. The average yield was 0.5μg/ml for non-amplified first round transformants.

[0051] Each pooled cell population was then cultured in the presence of10⁻⁷M MTX, and after two weeks, resistant colonies were again pooled andtitred for Campath-1H production. There was a considerable increase inyield of up to 80-fold (Table 1). These cells were dilution cloned,screened for Campath-1H yield, and two high producer lines isolated,called A37 and 3D9 (Table 1). These were both amplified further in thepresence of 10⁻⁶M MTX, then dilution cloned and screened as above. Theincrease in expression ac this second, and final, amplification stagewas not so dramatic as seen previously; nevertheless, when re-fed atconfluence and left for a further 4 days, the cell lines A39 and 3D11were capable of producing up to 200 μg/ml of Campath-1H. TABLE 1Expression Levels of Campath-1H using Stepwise Amplification AccumulatedConstruct Selection stage Campath-1H (μg/ml) pLD9 + pNH316 dhfr⁺/neobasal pool 0.5 10⁻⁷ M MTX amplified pool 18-40 Cell lines A37 and 3D9 4010⁻⁶ M MTX amplified pool 60-90 Cell line A39 100 Cell line 3D11 150-200

[0052] Legend to Table

[0053] Cells were allowed to reach confluence in a T-175 tissue cultureflask, then re-fed with fresh 50 ml of tissue culture medium and leftfor a further 4 days. The Campath-1H antibody that had accumulated inthe medium during this period was measured by ELISA. Total cell countson the day of assay were usually 2.5×10⁷. The yield from the 3D11 cellline reflects a productivity of 100 μg/10⁶ cells/day.

[0054] The co-transfection vectors pLD9 and pNH316 were further employedto evaluate an alternative amplification strategy to the one describedabove. The dhfr⁻ CHO cells were co-transfected as usual, and two dayslater split directly into a series of flasks containing G418 (forneomycin selection) and increasing concentrations of MTX ranging from3×10⁻⁹M to 10⁻⁷M. Following two weeks of this selection, the number ofresistant colonies were counted and pooled for each flask. When the cellpopulations had stabilized, they were assayed for Campath-1H antibodytitres and the results are shown in Table 2. As the MTX level wasincreased, there was a marked decrease in the number of surviving dhfr⁺colonies, but they expressed proportionately more Campath-1H. Thus, in aone step direct selection at high concentrations of MTX, it is possibleto isolate cell populations which produce up to 60-fold increase inantibody yield compared to cell populations selected for basal dhfrlevels. TABLE 2 Expression Levels of Campath-1H using Direct SelectionAccumulated Selection (M MTX) dhfr⁺ colonies Campath-1H (μg/ml) No MTX500 0.5 3 × 10⁻⁹ 40 2 10⁻⁸ 5 7 3 × 10⁻⁸ 5 30 10⁻⁷ — —

[0055] Legend to Table

[0056] Colonies at each MTX selection stage were pooled and assayed asdescribed in the legend of Table 1.

[0057] This selection procedure was repeated following anotherco-transfection of cells, and in this instance, the entire populationwas selected in medium containing G418 and 3×10⁻⁸M MTX. This generated alarger pool of resistant colonies which were subsequently pooled andre-amplified twice more using MTX concentrations of 6×10⁻⁷M, then3×10⁻⁶M. At this stage, the cells were dilution cloned and screened forCampath-1H levels. The two highest producer cell lines isolated werecapable of producing antibody levels up to 100-150 μg/ml and weredesignated as lines 4F11 and 5E10.

[0058] The growth rates of these cell lines, and the A39/3D11 linesdescribed above, were considerably slower than the parentalnon-transformed dhfr⁻ CHO cells. This is usually a common feature ofthese cells once they have been engineered to express high quantities ofa product gene. The yields from the 5E10 and 4F11 cell lines proved tobe quite variable over time, and the latter appeared to have only alimited passage life lasting about 3 weeks before entering crisis anddeath. This instability was not evident at all in the other cell lines,although in general, the lines isolated from the second amplificationprocedure, including 5E10, were usually more fickle to culture. Of allthe lines, the 3D11 coupled good growth and stability with highCampath-1H yields. To ensure the propagation of these features, the 3D11cell line was dilution cloned once more to generate the 3D11* line andthis similarly produced Campath-1H yields up to 200 μg/ml.

EXAMPLE 4 Growth of and Production from C1H 3D11* 44 in WCM4

[0059] a) C1H 3D11* cells growing as a monolayer in Iscoves +10% FBSFlow, non-essential amino acids, 10⁻⁶M Methotrexate and antibiotics wereapproximately 90% confluent. These cells were removed from the plasticwith trypsin/versene, washed in Iscoves medium without supplements,centrifuged and resuspended at 5×10⁴/ml in WCM4 medium Table 3+0.25%peptone +0.1% polyethylene glycol (PEG) 10,000+0.5% fetal boine serum(FBS) without methotrexate (MTX). TABLE 3 Formulation for medium WCM4Iscoves modification of DMEM without BSA, transferrin and lecithin.Available from GIBCO Ltd., Unit 4, Cowley Mill Td. Est., Uxbridge UB827G. Similar to published medium (Iscoves and Melcher (1978) J. Exp.Med. 1. 47, 923) without the bovine serum albumin, pure humantranferrin, or soyabean lecithin. +5 ml/liter 200 mM L glutamine +50mg/liter L proline +50 mg/liter L threonine +50 mg/liter L methionine+50 mg/liter L cysteine +50 mg/liter L tyrosine +25 mg.liter ascorbicacid +0.062 mg.liter vitamin B6 +1.36 mg.liter vitamin B12 +0.2 mg/literlipoic acid +0.088 mg/liter methyl linoleate +1 μM methotrexate +1mg/liter FeSO₄ +1 mg/liter ZnSO₄ +0.0025 mg/liter CuSO₄ +5 mg/literrecombinant insulin (Nucellin) +50,000 Iu/liter polymyxin +20,000Iu/liter neomycin +0.16 mg/liter putrescine-2 HCL.

[0060] Three 25 cm² flasks were set up with 10 ml of cell suspension+hypoxanthine (H), thymidine (T) or HT. These flasks were incubated at36.5° C. in 5% CO₂ incubator.

[0061] After six days, the flasks were pooled and added to an equalvolume of WCM4+MTX without peptone or PEG, and were transferred to a 75cm² flask.

[0062] These cells were used to seed a 500 ml Techner spinner, incubatedat 36.5° C. spinning at 40 rpm. Cells continued growing-serum free for aperiod of over five months and although it was found that the cellsneeded a period of adaptation, the growth rate and viability steadilyimproved. The population doubling time was calculated to be 73.1 hoursover approximately 7 weeks; this decreased to 47.4 hours over thesubsequent 20 days then stabilised. Antibody secretion remained high atlevels in excess of 60 μg/ml. It was determined that the gene copynumber in these cells did not decrease according to band intensity usingNorthern blot analysis.

[0063] In fermenters, these cells produced antibody in excess of 70μg/ml and regularly achieve levels of 100 μg/ml or more. These cells aredenoted C1H 3D11* 44.

[0064] b) Cells from a) above which had been growing serum-free for over2 months were transferred to a SGi 1 litre fermenter with a stainlesssteel angled paddle turning at 70 rpm. The temperature was set at 37°C., dO₂ at 10% and pH control to 7-7.2. The fermenter was seeded on day0 with 0.22×10⁶ cells/ml in WCM4 (Table 3) with 0.1% polyethylene glycol(PEG) 10,000 and 0.25% soy peptone, and was top gassed with O₂ The cellswere routinely passaged using fresh medium and a split rate typicallybetween 1 to 2 and 1 to 4.

[0065] On day 33 the top gassing was replaced with deep sparging whichis can be expected to cause more physical damage to the cells.

[0066] On day 50 onwards WCM5 (Table 4) was used together with peptoneand PEG instead of WCM4. TABLE 4 Formulation for Medium WCM5 Iscovesmodification of DMEM without BSA, transferrin or lecithin (see Table 3).+5 ml/liter 200 mM L glutamine +50 mg/liter L proline +50 mg/liter Lthreonine +50 mg/liter L methionine +50 mg/liter L cysteine +50 mg/literL tyrosine +25 mg/liter L ascorbic acid +0.062 mg.liter Vitamin B6 +1.36mg.liter Vitamin B12 +2 mg/liter Ferric citrate +1 mg/liter Zincsulphate +0.0025 mg.lit Copper sulphate +50,000 IU/liter Polymyxin+20,000 IU/liter Neomycin +3 μl/liter Ethanolamine +0.16 mg/literPutrescine +5 mg/liter Recombinant Insulin (Nucellin)

[0067] On day 53 the PEG was replaced with 0.1% pluronic F68. Theresulting growth and antibody levels achieved are shown the the attachedgraphs (FIGS. 4 and 5), and demonstrate the capacity of the invention toallow protein-free production of antibody in excess of 100 μg/ml infermenters.

EXAMPLE 5 Analysis of the Rate of Campath-1H Synthesis and Secretionfrom CHO Cells

[0068] During the course of culturing the Campath-1H producing CHO cellsof Example 3, it became clear that even when they reached confluence,antibody levels continued to accumulate, with time, in the culturemedium. To determine whether this was possibly a consequence ofintracellular accumulation coupled to slow secretion, the rates ofCampath-1H synthesis and secretion were measured using A39 cells. Theseanalyses were performed over 3-4 consecutive days on cells which wereeither in growth phase, or confluent stationary phase. For the cells ineither growth state, the results were identical, and data is presentedonly for the immuno-precipitated radiolabelled Campath-1H produced fromstationary cells.

[0069] The rate of antibody synthesis was measured by pulsing the cellsfor a short period with [S³⁵]-methionine on each of four consecutivedays, and then examining the quantity, and distribution, ofimmuno-precipitated material. In FIG. 2, it is clear that the rate ofsynthesis is equally high at all time points measured. Furthermore, evenby the end of this short pulse, in each case, more than half of thenewly synthesized Campath-1H is already present in the medium suggestingrapid secretion. This was confirmed by the data shown in FIG. 3, inwhich parallel cells were similarly pulsed, and the distribution of theradiolabelled Campath-1H chased over a three day period. Within 24hours, virtually all of the cellular radiolabelled antibody has beenchased into the medium, where it remained stable for the duration of theexperiment. This demonstrates that even when the recombinant CHO cellsremain stationary for long periods, the rates of Campath-1H synthesisand secretion are not diminished.

[0070] Campath-1H ELISA assay. Microtiter plates were coated withanti-human IgG and incubated with the assay sample (in culture medium).Antibody detection was visualized by using an anti-human gamma chainspecific peroxidase conjugate.

[0071] Analysis of rates of Campath-1H synthesis and secretion. Cellsfrom Example 3 were grown to confluence in 3 cm tissue culture wells,then incubated for 30 minutes in methionine-free Dulbeccos's MEMcontaining 10% fetal calf serum. Following this, the cells were labelledin the presence of 120 μCi/ml [³⁵S] methionine (>800Ci/mmol; Amersham)for the appropriate time period, then either harvested and lysed in 500μl of NP-40 lysis buffer, or incubated further in normal growth medium.Then 125 μl aliquots of cell lysate or culture medium wereimmuno-precipitated using goat anti-human IgG (heavy chain specific;Sigma) and 10 % protein-A Sepharose (Pharmacia). Samples were thenseparated on 10% SDS-PAGE reducing gels according to Laemmli and thesignals amplified with Enhance (NEN-Dupont). The dried gels were thenautoradiographed overnight.

[0072] Biological Assays for Functional CHO-glycosylated Campath 1H

[0073] Complement Lysis Assay for Campath 1H

[0074] The complement lysis assay is a measure of antibody functionexpressed as specific activity, determined by the ability of aCHO-glycosylated antibody of known concentration to bind to apre-determined number of cells and effect cell lysis.

[0075] The assay is carried out on Campath 1H from Example 4 usingKarpas 422 cells (established from B-cell non-Hodgkin lymphoma cellline—Dyer et al., (1990) Blood, 75 704-714) expressing Campath antigenon the cell surface. 1.2×10⁷ cells were loaded with radiolabel byincubating for 2 hours at 37° C. in a CO₂ incubator in the presence of600 μCi of 51Cr (sodium chromate).

[0076] 5.3 ml of the loaded cells in medium (total volume 23.5 ml), wereadded to 12.5 ml of normal human serum and 150 μl of the mixture werepipetted into the wells of a microtitre plate.

[0077] 50 μl samples of the final eluate from three purification runswere mixed with the cells and incubated for 30 minutes at 4° C. followedby 90 minutes at 37° C. The culture was centrifuged at 2000 rpm for 5minutes and the radioactivity in 100 μl of cell supernatant was countedon a gamma counter. Complement lysis activity in Kilo Units/ml wascalculated from a standard curve of a reference preparation (1000Units/ml).

[0078] The results are set out in Table 5.

[0079] The concentration of Campath 1H in the 50 μl samples of finaleluate was estimated using samples in PBS pH 7.2 read on aspectrophotometer at 280 nm. The results are expressed in Table 3 asoptical density in mg/ml.

[0080] From this data the specific activity:$\frac{{KU}\text{/}{ml}}{OD}$

[0081] is determined. TABLE 5 Complement lysis Protein Conc SpecificSample Kilo Units/ml mg/ml Activity A 11.2 11.1 1.0 B 14.8 14.2 1.0 C13.7 13.6 1.0

[0082] The results indicate that CHO-glycosylated Campath 1H isfunctional.

[0083] Treatment of an Individual with CHO-glycosylated Campath 1H

[0084] An individual diagnosed as having severe T-cell mediatedinflammation of the joints (immobilising polyarthritis, pleuritis,abdominal pains) over five years requiring long periods ofhospitalisation was treated with CHO derived Campath 1H from Example 4using the following regime:

[0085] 2 mg per day over 6 days by intravenous injection

[0086] 10 mg per day over subsequent 6 days by intravenous injection.

[0087] During the second 6 day treatment there was a significantsymptomatic improvement. By the end of the second period the jointinflammation was much improved and a skin abscess had cleared withantibiotic treatment. Thirty days after the end of the treatment theindividual was discharged.

[0088] Approximately 9 months after the initial treatment, theindividual suffered a relapse with multiple joint involvement. Afterinitial testing for sensitivity with a low dose, the individual wasgiven a further course of treatment with 10 mg/day Campath 1H for 10days with significant improvement.

EXAMPLE 6

[0089] Expression of Humanised ANTI-CD4 Antibody from Cho Cells

[0090] Construction of the Expression Vector pBan1: Modification of2342-12

[0091] The complementarily determining regions from a rat IgG2b raisedagainst human CD4 (The New England Journal of Medicine 1990 323:250-254) were grafted onto human heavy and light chain frameworks(Winter et al, Nature, 1988, 322 323-327).

[0092] The cDNA encoding the humanised CD4 light chain was cloned intopLD9 (Page and Sydenham, M. A. 1991 Biotechnology 9 64-681. Theresulting plasmid was designated p2110. The humanised CD4 heavy chainwas sequenced and cloned into a modified version of plasmid p342-12 [LawM-F., Byrne, J. C. and Hinley, P. M. 1983 Mol. Cell. Biol. 3 2110-2115).

[0093] Plasmid p342-12 was digested with BamH1 to remove the 7.4 kbpfragment containing part of the BPV-1 genome. The backbone containingthe β-lactamase gene and the neomycin resistance gene under the controlof the mouse metallothionine promoter was purified and religated at theBamH1 site. This plasmid was digested with HinDIII, incubated with thelarge fragment of DNA polymerase I to remove the HinDIII site and thenrelegated. The β-actin expression cassette, containing the β-actinpromoter immediately upstream of a unique HinDIII site followed by thepolyadenylation signal, was cloned into the BamHI site of the modifiedp342-12 plasmid to generate pban1.

[0094] Plasmid pBan1, therefore, consisted of the neomycin resistancegene, the β-lactamase gene and the β-actin expression cassettecontaining the unique HinDIII site. The cDNA encoding the humanisedheavy chain was cloned into this site and the resulting plasmidcontaining the correctly orientated insert was designated pBanCD4H.Thus, p2110 and pBanCD4H contained a different selectable marker andco-transfection into recipient dhfr-CHO cells would permit the directselection and isolation of dhfr⁺/neo^(r) colonies. Cells exhibiting thisphenotype should express functional antiCD4 antibody and could beamplified to elevate the antibody titres.

[0095] Expression of anti-CD4 antibody in CHO cells

[0096] a) Cell culture methods.

[0097] The dhfr—CHO line DUK-B11 [Urlaub, C. and Chasin, L. A. 1980Proc. Natl. Acad. Sci. USA 77 4216-4220] was propagated in Iscoves MEMmedium supplemented with 10% foetal bovine serum and ⁴ μg each ofhypoxanthine and thymidine (all Flow). After transfection, transformantswere selected in the medium described above except that thehypoxanthine/thymidine were omitted and dialysed foetal bovine serum wasused. In addition, G418 was included at 500 μg/ml. To induce spontaneousamplification of sequences containing and flanking the dhfr gene, MTXwas added to a concentration of 0.1 μM.

[0098] b) Transfection and amplification

[0099] The dhfr—CHO cell line DUK-B11 was co-transfected with 5 μg ofp2110 and 5 μg of pBanCD4H using the transfectam reagent under theconditions recommended by the manufacturer. Transformants were selectedfor the dhfr⁺/neo^(r) phenotype as described above. Several hundreds oftransformants were observed and pooled. Initital titres indicated thatthe first round basal transformants were secreting about 0.1 μg/ml/day.This po led population was then cultured in the presence of 0.1 μM MTXfor about 14 days. Resistant colonies were again pooled and assayed.Expression had increased some 100 fold, the pooled, amplified coloniesproducing about 10-12;1g/ml/day. In order to obtain stable, clonal celllines giving high antibody titres, the resistant pools were cloned bylimiting dilution in 96-well plates. Fifty single colonies wereidentified and assayed and the four lines giving the highest titrespropagated. This process of identifying highly expressing clones withinthe resistant population produced a line designated D419 which expressedthe anti-CD4 antibody at about 20 μg/ml/day.

[0100] Characterisation of dhfr⁺/neo^(r) cell lines

[0101] i) Determination of copy number and steady state transcriptionlevels by slot blot analysis of DNA and RNA.

[0102] Whole cell RNA and DNA was prepared from the various stages ofamplification as described by Maniatis et al. [1982 Molecular Cloning. ALaboratory Manual. Cold Spring Harbour Lab ratery, Cold Spring Harbour,N.Y.]. After fixing onto nitrocellulose filters, the nucleic acids wereprobed with [³²-P]-αATP labelled DNA sequences of the heavy chain, thedhfr gene and the β-actin gene as a control “housekeeping” gene toeliminate artifacts due to loading errors.

[0103] Inititally, the uncloned 0.1 μM MTX amplified pool was comparedto the first round unamplified transformants and the untransformedparental B11 cells, with the probes described. Accordingly, no DNAsignal was detected in the parental line when probed with the heavychain but a weak signal was detected for dhfr. This is due to thesingle, non-functional dhfr allele in the B11 cell line. As a result, noRNA signal was detected with either probe. In contrast, a strong signalwas detected with both probes on RNA and DNA in the primarytransformants which reflects the start of expression. A very significantincrease in copy number and steady state levels of RNA of heavy chainand dhfr is observed in the uncloned amplified pool. This accuratelycorrelates with the observed increase in expression. Steady state levelsof β-actin RNA were consistent in all three lines examined.

[0104] A similar comparison was made between the four highest expressingcloned cell lines. A strong signal was detected on both the RNA and theDNA blots. However, although the DA19 line was expressing twice as muchantibody as a line designated D423, this difference was not in eitherthe copy number or steady state levels of RNA. There are two possibleexplanations for this observation; the first is that the DNA in the DA19line has integrated at a site in the genome at which it is under theinfluence of an enhancer. However, this presumably would be reflected inelevated levels of RNA. The more likely explanation is that in thereplication and duplication of the tandem arrays in the line D423, someof the copies of the dhfr/antibody cassette have undergonere-arrangement and are non-functional and truncated. This is notuncommon since the site of integration of heterologous genes is often atbreakpoints in the chromosomes such as telomeres which are known to be“hot spots” for such re-arrangements. This could be resolved by Northernand Southern analysis.

[0105] ii) Protein synthesis and secretion of anti-CD4 antibody in theD419 line

[0106] The clonal D419 line was labelled with ³⁵S-methionine andcysteine and the intracellular and secreted antibody extracted byimmunoprecipitation with appropriate antibodies. Followingelectrophoresis on reducing SDS-PAGE gels, the gels were dried and thesignal detected by autoradiography.

[0107] It was clear from the result chat both heavy and light chain areefficiently synthesised. Intracellularly, there need not be stochiometrybetween heavy and light chains since the two associate as they passthrough the secretory organelles. However, close stochiometry isobserved in the secreted material.

1. A method of treating a human patient suffering from a disease ordisorder responsive to treatment with a therapeutic antibody, whichcomprises repeated administration of a therapeutically effective amountof a human or humanized antibody expressed and glycosylated in a CHOcell expression system, to a patient in need thereof.
 2. The method ofclaim 1, wherein said disease or disorder is selected from the groupconsisting of T-cell mediated disorders, autoimmune disorders, cancerand infections diseases.
 3. The method of claim 2, wherein said T-cellmediated disease is selected from the group consisting of severevasculitis, rheumatoid arthritis and systemic lupus.
 4. The method ofclaim 2, wherein said autoimmune disorder is selected from the groupconsisting of multiple sclerosis, graft versus host disease, psoriarsis,juvenile onset diabetes, Sjogrens' disease, thyroid disease, myastheniagravis, transplant rejection and asthma.
 5. The method of claim 2,wherein said cancer is non-Hodgkin's lymphoma or multiple myeloma. 6.The method of claim 2, wherein said infectious disease is HIV or herpes.7. The method of claim 1, wherein the antibody is administered daily forup to 30 days.
 8. The method of claim 7, wherein the dose of antibody isbetween 1 to 100 mg.
 9. A method of treating a human patient sufferingfrom a disease or disorder responsive to treatment with a therapeuticantibody, which comprises administering a therapeutically effectiveamount of a human or humanized antibody expressed and glycosylated in aCHO cell expression system, to a patient in need thereof, in a two-partdosing regime, wherein the antibody is administered in different dosesin each part of the two-part dosing regime.
 10. The method of claim 9,wherein the two-part dosing regime comprises a first dosing regime, inwhich the antibody is administered at a dose of 1 to 5 mg for 5-10 daysand a second part, in which the antibody is administered at 6-15 mg foran additional 5-10 days.